Isolation And Characterization Of Human Genes Affecting Chromosome Metabolism

National Institute Of Environmental Health Sciences

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Abstract

p53 MASTER REGULATORY NETWORK TRANSACTIVATION . The DNA binding activity of the master regulator p53 is critical to its tumor suppressor activity in response to cellular and environmental stresses. The role of binding to individual response elements (REs) in transactivation specificity was investigated using a highly regulatable p53 assay in yeast and human cells. Nearly all REs have at least 1 mismatch from the 20 base consensus sequence and spacers tend to consist of only a few bases at most. We are deconstructing the canonical p53 consensus sequence in order to understand the role of sequence, organization and level of p53 on transactivation in budding yeast and human cell systems. Contrary to early reports for in vitro binding, increases in spacer length of only a few bases greatly reduces p53 transactivation. While p53 lacked transactivation capacity from many full-sized RE canonical sequences, it functioned to different extents from several noncanonical sites that are frequent in the genome including 3/4 and 1/2 sites. Surprisingly, there can be substantial transactivation even at half-sites depending on sequence and p53 expression level. Efficient transactivation from canonical and noncanonical elements requires tetrameric p53. Thus, RE sequence and organization can have a large impact on p53-mediated transactivation. Furthermore, the functionality of noncanonical sequences greatly expands the p53 transcriptional network. In collaboration with the Dr. Douglas Bell group we have been involved in the development of a high-throughput genomics system using cellular extracts and a microsphere assay that detects protein-DNA binding (MAPD) for elucidating functional impacts of p53 protein and RE sequence variation and spacers in transcriptional networks.[unreadable] FUNCTIONALITY OF HUMAN p53 MUTANTS. p53 missense mutations in the DNA binding domain are often cancer-associated. As shown with yeast-based systems, p53 mutants can alter spectrum and intensity of transactivation from individual REs. We addressed in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV, ionizing radiation or spontaneously. These patterns reflected changes in the spectra and activity of p53 target genes and showed that p53 mutations in human cells can differentially influence target gene transactivation resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy. We are also investigating how WT and mutant p53 can regulate genes through novel (noncanonical) sequence. [unreadable] EVOLUTION OF p53 TRANSCRIPTIONAL NETWORK. Networks can evolve through variation of master regulators and/or by changes in regulation of genes within networks. Using a combination of custom bioinformatics and multispecies alignment of promoter regions, we investigated the functional evolution of REs in terms of responsiveness to the sequence-specific transcription factor p53. We identified REs orthologous to known p53 targets in human and rodent cells or alternatively REs related to the established p53 consensus. The orthologous REs were assigned p53 transactivation capabilities based on rules determined from model systems, and a functional heat map was developed for p53 transactivation towards 38 genes in 14 species. This approach emphasizes not only conservation of functionality but also addresses conservation of level of responsiveness. Individual REs exhibited marked differences in potential transactivation as well as widespread turnover of functional binding sites during p53 network evolution. Functional differences were often not predicted from consensus sequence evaluations. Of the 43 established human p53 REs analyzed, 50% were nonfunctional in rodents. Surprisingly, there was almost no conservation of functional REs or compensatory REs for genes involved in DNA metabolism or repair suggesting important differences in p53 stress responses as well as cancer development between humans and rodents. Currently our efforts are directed to evaluating the conservation of noncanonical p53REs as well as spacer length.[unreadable] VARIATION WITHIN THE p53 NETWORK. We had developed a system to identify SNPs in potential p53 REs that are predicted to modify p53 control of target genes and establish their functional transactivation consequences. Recently we characterized a SNP in the promoter of the angiogenic factor FLT-1 receptor that might result in p53 transactivation. Interestingly the isolated p53 RE, which was actually a half-site, did not function alone and its transactivation required another upstream site that corresponded to an estrogen receptor half-site. The presence of both functional REs resulted in synergistic transcriptional activation of FLT1. Following these findings we had used the FLT1 promoter as a model, to understand in detail the synergistic mechanism of ERE with different p53 REs and in response to several environmental stresses.[unreadable] EXPANSION OF p53 TRANSCRIPTIONAL NETWORK. In a more global sense, these findings dramatically increase the size of a master regulatory network because half-sites contribute to regulation, and they demonstrate new opportunities for cross-talk between networks. Using the p53 transactivation rules in combination with bioinformatic approaches we are employing genome-wide approaches to determine other putative p53 target genes containing p53 half-site REs that could present similar regulation. In a preliminary search 15 novel putative p53 target genes were identified including some p53REs. We demonstrated that the RAP80 gene, involved in the DNA damaging signaling, is indeed a p53 target gene. In addition we identified a link between these two proteins (RAP80 and p53) and showed that they are part of a novel auto-regulatory loop consisting of RAP80, HDM2 and the p53. This loop includes transcriptional regulation of the RAP80 gene by p53 through a noncanonical p53 target sequence, interaction of RAP80 with p53 and HDM2, and negative modulation of p53 stability and activity by RAP80. The second gene analyzed is toll-like receptor 3 (TLR3) involved in the innate immune pathway and detection of invading pathogens particularly double-stranded RNA viruses. We demonstrated that different DNA damaging agents which activate p53 resulted in TLR3 gene activation. We also observed that p53 binds the RE identified in the promoter region of TLR3 under these stresses. In addition we have also identified and established that the promoters of several Toll receptors have canonical and noncanonical p53 target REs as well as SNPs in the target REs.[unreadable] CLINICAL IMPLICATIONS. The systems developed in yeast have provided opportunities to address the functional consequences of p53 mutations identified in tumors. As part of a screening of patients undergoing neoadjuvant treatment for breast cancer, p53 mutations are identified. The mutations are then characterized in terms of transcriptional function and related to disease characteristics. [unreadable] FRATAXIN. Friedreichs ataxia is a quantitative disease in that the level of reduction of frataxin appears to correlate with disease severity. Previously we had found with a yeast system that reduced levels of frataxin led to a) iron accumulation within mitochondria; b) mitochondrial DNA damage/loss and protein damage; and c) nuclear damage. To assess the biological consequences of reduced frataxin levels in human cells, we are utilizing RNA interference to lower frataxin levels 70 to 90%, similar to levels in FRDA. Preliminary results indicate that reduced levels of frataxin results in much high levels of mitochondrial lesions following treatment with ROS (hydrogen peroxide).